Isabelle Seif

Lack of Barrels in the Somatosensory Cortex of Monoamine Oxidase A–Deficient Mice: Role of a Serotonin Excess during the Critical Period

In a transgenic mouse line (Tg8) deficient for the gene encoding monoamine oxidase A (MAOA), we show that the primary somatosensory cortex (S1) lacks the characteristic barrel-like clustering of layer IV neurons, whereas normal pattern formation exists in the thalamus and the trigeminal nuclei. No barrel-like patterns were visible with tenascin or serotonin immunostaining or with labeling of thalamocortical axons. An excess of brain serotonin during the critical period of barrel formation appears to have a causal role in these cortical abnormalities, since early administration of parachlorophenylalanine, an inhibitor of serotonin synthesis, in Tg8 pups restored the formation of barrels in S1, whereas inhibition of catecholamine synthesis did not. Transient inactivation of MAOA in normal newborns reproduced a barrelless phenotype in parts of S1.

Type I IFN as a Natural Adjuvant for a Protective Immune Response: Lessons from the Influenza Vaccine Model

The identification of natural adjuvants capable of selectively promoting an efficient immune response against infectious agents would represent an important advance in immunology, with direct implications for vaccine development, whose progress is generally hampered by the difficulties in defining powerful synthetic adjuvants suitable for clinical use. Here, we demonstrate that endogenous type I IFN is necessary for the Th1 type of immune response induced by typical adjuvants in mice and that IFN itself is an unexpectedly powerful adjuvant when administered with the human influenza vaccine, for inducing IgG2a and IgA production and conferring protection from virus challenge. The finding that these cytokines, currently used in patients, are necessary for full expression of adjuvant activity and are sufficient for the generation of a protective immune response opens new perspectives in understanding the basis of immunity and in vaccine development.

Gap Junction-Mediated Astrocytic Networks in the Mouse Barrel Cortex

The barrel field of the somatosensory cortex constitutes a well documented example of anatomofunctional compartmentalization and activity-dependent interaction between neurons and astrocytes. In astrocytes, intercellular communication through gap junction channels composed by connexin 43 and 30 underlies a network organization. Immunohistochemical and electrophysiological experiments were undertaken to determine the coupling properties of astrocyte networks in layer IV of the developing barrel cortex. The expression of both connexins was found to be enriched within barrels compared with septa and other cortical layers. Combination of dye-coupling experiments performed with biocytin and immunostaining with specific cell markers demonstrated that astrocytic networks do not involve neurons, oligodendrocytes or NG2 cells. The shape of dye coupling was oval in the barrel cortex whereas it was circular in layer IV outside the barrel field. Two-dimensional analysis of these coupling areas indicated that gap junctional communication was restricted from a barrel to its neighbor. Such enrichment of connexin expression and transversal restriction were not observed in a transgenic mouse lacking the barrel organization, whereas they were both observed in a double-transgenic mouse with restored barrels. Direct observation of sulforhodamine B spread indicated that astrocytes located between two barrels were either weakly or not coupled, whereas coupling within a barrel was oriented toward its center. These observations indicated a preferential orientation of coupling inside the barrels resulting from subpopulations of astrocytes with different coupling properties that contribute to shaping astrocytic networks. Such properties confine intercellular communication in astrocytes within a defined barrel as previously reported for excitatory neuronal circuits.

Oxidative Stress–Dependent Sphingosine Kinase-1 Inhibition Mediates Monoamine Oxidase A–Associated Cardiac Cell Apoptosis

The mitochondrial enzyme monoamine oxidase (MAO), its isoform MAO-A, plays a major role in reactive oxygen species–dependent cardiomyocyte apoptosis and postischemic cardiac damage. In the current study, we investigated whether sphingolipid metabolism can account for mediating MAO-A– and reactive oxygen species–dependent cardiomyocyte apoptosis. In H9c2 cardiomyoblasts, MAO-A–dependent reactive oxygen species generation led to mitochondria-mediated apoptosis, along with sphingosine kinase-1 (SphK1) inhibition. These phenomena were associated with generation of proapoptotic ceramide and decrease in prosurvival sphingosine 1-phosphate. These events were mimicked by inhibition of SphK1 with either pharmacological inhibitor or small interfering RNA, as well as by extracellular addition of C2-ceramide or H2O2. In contrast, enforced expression of SphK1 protected H9c2 cells from serotonin- or H2O2-induced apoptosis. Analysis of cardiac tissues from wild-type mice subjected to ischemia/reperfusion revealed significant upregulation of ceramide and inhibition of SphK1. It is noteworthy that SphK1 inhibition, ceramide accumulation, and concomitantly infarct size and cardiomyocyte apoptosis were significantly decreased in MAO-A–deficient animals. In conclusion, we show for the first time that the upregulation of ceramide/sphingosine 1-phosphate ratio is a critical event in MAO-A–mediated cardiac cell apoptosis. In addition, we provide the first evidence linking generation of reactive oxygen species with SphK1 inhibition. Finally, we propose sphingolipid metabolites as key mediators of postischemic/reperfusion cardiac injury.

Effects of Monoamine Oxidase A Inhibition on Barrel Formation in the Mouse Somatosensory Cortex: Determination of a Sensitive Developmental Period

Genetic inactivation of monoamine oxidase A (MAOA) in C3H/HeJ mice causes a complete absence of barrels in the somatosensory cortex, and similar alterations are caused by pharmacological inhibition of MAOA in wild type mice. To determine when and how MAOA inhibition affects the development of the barrel field, the MAOA inhibitor clorgyline was administered to mice of the outbred strain OF1 for various time periods between embryonic day 15 (E15) and postnatal day 7 (P7), and the barrel fields were analyzed with cytochrome oxidase and Nissl stains in P10 and adult mice. High‐pressure liquid chromatography measures of brain serotonin (5‐HT) showed three‐ to eightfold increases during the periods of clorgyline administration. Perinatal mortality was increased and weight gain was slowed between P3 and P6. Clorgyline treatments from E15 to P7 or from P0 to P7 disrupted the formation of barrels in the anterior snout representation and in parts of the posteromedial barrel subfield (PMBSF). Treatments from P0 to P4 caused similar although less severe barrel field alterations. Clorgyline treatments only during embryonic life or starting on P4 caused no detectable abnormalities. In cases with barrel field alterations, a rostral‐to‐caudal gradient of changes was noted: Rostral barrels of the PMBSF were most frequently fused and displayed an increased size tangentially.

Altered regulation of the 5-HT system in the brain of MAO-A knock-out mice

Genetic deficiency of monoamine oxidase‐A (MAO‐A) induces major alterations of mood and behaviour in human. Because serotonin (5‐HT) is involved in mood regulation, and MAO‐A is responsible for the catabolism of 5‐HT, we investigated 5‐HT mechanisms in knock‐out mice (2‐month‐old) lacking MAO‐A, using microdialysis, electrophysiological, autoradiographic and molecular biology approaches. Compared to paired wild‐type mice, basal extracellular 5‐HT levels were increased in ventral hippocampus (+202%), frontal cortex (+96%) and dorsal raphe nucleus (DRN, +147%) of MAO‐A mutant mice. Conversely, spontaneous firing rate of 5‐HT neurons in the DRN (recorded under chloral hydrate anaesthesia) was ∼40% lower in mutants. Acute 5‐HT reuptake blockade by citalopram (0.2 and 0.8 mg/kg i.v.) produced a much larger increase in extracellular 5‐HT levels (by ∼4 fold) and decrease in DRN neuronal firing (with a ∼4.5 fold decrease in the drugs ED50) in MAO‐A knock‐out mice, which expressed lower levels of the 5‐HT transporter throughout the brain (−13 to −34% compared to wild‐type levels). The potency of the 5‐HT1A agonist 8‐OH‐DPAT to produce hypothermia and to reduce the firing of DRN serotoninergic neurons was significantly less in the mutants, indicating a desensitization of 5‐HT1A autoreceptors. This was associated with a decreased autoradiographic labelling of these receptors (−27%) in the DRN. Altogether, these data indicate that, in MAO‐A knock‐out mice, the enhancement of extracellular 5‐HT levels induces a down‐regulation of the 5‐HT transporter, and a desensitization of 5‐HT1A autoreceptors which allows the maintenance of tonic activity of 5‐HT neurons in the DRN.

Developmental expression of monoamine oxidases A and B in the central and peripheral nervous systems of the mouse

Monoamine oxidases A (MAOA) and B (MAOB) are key players in the inactivation pathway of biogenic amines. Their cellular localization has been well established in the mature brain, but nothing is known concerning the localization of both enzymes during development. We have combined in situ hybridization and histochemistry to localize MAOA and MAOB in the developing nervous system of mice. Our observations can be summarized as five key features. (1) MAOA is tightly linked to catecholaminergic traits. MAOA is expressed in all noradrenergic and adrenergic neurons early on, and in several dopaminergic cell groups such as the substantia nigra. MAOA is also expressed in all the neurons that display a transient tyrosine hydroxylase expression in the brainstem and the amygdala and in neurons with transient dopamine‐β‐hydroxylase expression in the cranial sensory ganglia. (2) MAOA and MAOB are coexpressed in the serotoninergic neurons of the raphe from E12 to P7. During postnatal life, MAOA expression declines, whereas MAOB expression remains stable. (3) MAOA is transiently expressed in the cholinergic motor nuclei of the hindbrain, and MAOB is expressed in the forebrain cholinergic neurons. (4) MAOA‐ and MAOB‐expressing neurons are also detected in structures that do not contain aminergic neurons, such as the thalamus, hippocampus, and claustrum. (5) Starting at birth, MAOB expression is found in a variety of nonneuronal cells, the choroid plexus, the ependyma, and astrocytes. These localizations are of importance for understanding the effects of monoaminergic transmission during development. J. Comp. Neurol. 442:331–347, 2002.

Blockade of substance P (neurokinin 1) receptors enhances extracellular serotonin when combined with a selective serotonin reuptake inhibitor: an in vivo microdialysis study in mice

Substance P antagonists of the neurokinin‐1 receptor type (NK1) are gaining growing interest as new antidepressant therapies. It has been postulated that these drugs exert this putative therapeutic effect without direct interactions with serotonin (5‐HT) neurones. Our recent microdialysis experiment performed in NK1 receptor knockout mice suggested evidence of changes in 5‐HT neuronal function ( Froger et al. 2001 ). The aim of the present study was to evaluate the effects of coadministration of the selective 5‐HT reuptake inhibitor (SSRI) paroxetine with a NK1 receptor antagonist (GR205171 or L733060), given either intraperitoneally (i.p.) or locally into the dorsal raphe nucleus, on extracellular levels of 5‐HT ([5‐HT]ext) in the frontal cortex and the dorsal raphe nucleus using in vivo microdialysis in awake, freely moving mice. The systemic or intraraphe administration of a NK1 receptor antagonist did not change basal cortical [5‐HT]ext in mice. A single systemic dose of paroxetine (4 mg/kg; i.p.) resulted in a statistically significant increase in [5‐HT]ext with a larger extent in the dorsal raphe nucleus (+ 138% over basal AUC values), than in the frontal cortex (+ 52% over basal AUC values). Co‐administration of paroxetine (4 mg/kg; i.p.) with the NK1 receptor antagonists, GR205171 (30 mg/kg; i.p.) or L733060 (40 mg/kg; i.p.), potentiated the effects of paroxetine on cortical [5‐HT]ext in wild‐type mice, whereas GR205171 (30 mg/kg; i.p.) had no effect on paroxetine‐induced increase in cortical [5‐HT]ext in NK1 receptor knock‐out mice. When GR205171 (300 µmol/L) was perfused by ‘reverse microdialysis’ into the dorsal raphe nucleus, it potentiated the effects of paroxetine on cortical [5‐HT]ext, and inhibited paroxetine‐induced increase in [5‐HT]ext in the dorsal raphe nucleus. Finally, in mice whose 5‐HT transporters were first blocked by a local perfusion of 1 µmol/L of citalopram into the frontal cortex, a single dose of paroxetine (4 mg/kg i.p.) decreased cortical 5‐HT release, and GR205171 (30 mg/kg i.p.) reversed this effect. The present findings suggest that NK1 receptor antagonists, when combined with a SSRI, augment 5‐HT release by modulating substance P/5–HT interactions in the dorsal raphe nucleus.

Ketanserin and tetrabenazine abolish aggression in mice lacking monoamine oxidase A

Mice deficient in monoamine oxidase A (MAO A) have elevated brain levels of 5-HT and manifest enhanced aggression. We used these mice as a model to study the role of 5-HT in aggression. Our results show that ketanserin and tetrabenazine (TBZ) strikingly abolished the aggressive behavior of MAO A-deficient mice. The anti-aggressive effect of ketanserin may be primarily mediated by 5-HT(2A) receptors. Another specific 5-HT(2A) antagonist, [R-(+)-a-(2, 3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methan ol (MDL 100907), also blocks the aggression of mutant mice but was less dramatic. Ketanserin and TBZ are both antagonists of the vesicular monoamine transporter (VMAT2). The anti-aggressive effect of TBZ and part of the effect of ketanserin may be mediated by the VMAT2. Using radioligand binding and autoradiography, we also showed that the numbers of VMAT2, 5-HT(1A), 5-HT(2A) and 5-HT(2C) sites are decreased in brains of mutant mice, which may reflect down-regulation by excess 5-HT. This study suggests that ketanserin and TBZ may be developed as novel anti-aggressive agents.

Biochemical, behavioral, physiologic, and neurodevelopmental changes in mice deficient in monoamine oxidase A or B

The availability of mutant mice that lack either MAO A or MAO B has created unique profiles in the central and peripheral availability of serotonin, norepinephrine, dopamine, and phenylethylamine. This paper summarizes some of the current known phenotypic findings in MAO A knock-out mice and contrast these with those of MAO B knock-out mice. Differences are discussed in relation to the biochemical, behavioral, and physiologic changes investigated to date, as well as the role played by redundancy mechanisms, adaptational responses, and alterations in neurodevelopment.